Many of the adhesins of Gram-negative bacteria are incorporated into heteropolymeric fibers assembled by the chaperone/usher pathway. These fibers range in morphology from composite pili assembled by members of the FGS (F1G1 short) subfamily of chaperones, to fibrous capsule-like structures assembled by the FGL (F1G1 long) subfamily of chaperones. Here we propose to build on our work on P and type 1 pili, which has provided a paradigm for the assembly of hundreds of virulence fibers in diverse Gram-negative organisms. Using biochemistry, genetics, electron microscopy and X-ray crystallography, we have defined the structure and mechanism of action of the periplasmic chaperones in significant detail. We have solved the structure of the PapD periplasmic chaperone in complex with the tip adaptor subunit, PapK, and with the major tip subunit, PapE. We have also solved the structure of the PapE subunit in complex with the N-terminal extension of PapK and the structure of the receptor binding domain of the PapG adhesin with and without its host carbohydrate ligand. Pilus chaperones are comprised of two immunoglobulin (Ig)-like domains. These structures showed that pilin subunits also have an Ig-like fold, but they are missing their seventh (G) strand, thus exposing the hydrophobic core. In a process we termed donor-strand complementation, the chaperone's G1 strand serves as the pilin's seventh strand, catalyzing the folding of the subunit and preventing non-productive subunit aggregation. At the usher, pilus assembly occurs by donor-strand exchange, in which the G1 strand of the chaperone is replaced by an N-terminal extension that is present on every subunit and exposed on incoming chaperone-subunit complexes. The pilus subunit then undergoes a topological transition that triggers the closure of its groove, cementing its neighbor's NTE as part of its own Ig fold. Thus, the final pilus structure is a series of Ig-like domains, each of which is formed from parts of two individual subunit monomers. Each subunit has distinct specificity for other interactive subunits and distinct roles in pilus assembly. We propose to expand our knowledge of the structures of the pilus adhesins and further study the structure of the subunits and assembly machinery of two such assembly systems: the prototypical FGS P pilus system of uropathogenic Escherichia coli and the Saf FGL system of Salmonella. In addition, we will perform structural studies on anti-chaperone compounds.